The invention relates to the area of transduction of extracellular signals into the nucleus of human cells. More particularly, the invention relates to transduction of extracellular signals by means of mitogen-activated protein kinase kinase pathways.
The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular cues into intracellular responses. MAPKs phosphorylate substrates on serine or threonine residues adjacent to proline residues and are thus proline-directed protein kinases, as described in Cano and Mahadevan, TIBS Reviews 20:117-122 (1995). It is believed that multiple MAPK cascades exist, thus implicating many other molecules in the up- and downstream events contributing to MAPK signal transduction events.
The c-Jun NH2-terminal kinase (JNK), or stress-activated protein kinase (SAPK), signal transduction pathway is activated in response to cellular stresses, including DNA damage, heat shock, and UV light, as well as by proinflammatory cytokines, such as TNFxcex1. (Gupta et al., 1996, EMBO J. 15:2760-70; Derijard et al., 1994, Cell 76:1025-37; Kyriakis et al., 1994, Nature 369:156-60; and Kallunki et al., 1994, Genes Dev. 8:2996-3007). After activation, JNK binds to and phosphorylates transcription factors such as ATF2 and c-Jun, which increases transcription of genes activated by these factors. Whitmarsh et al., 1996, J. Mol. Med. 74:589-607.
The pathways responsible for transducing environmental stress signals through JNK have not been fully described. Thus, there is a need in the art for the identification of proteins which are involved in JNK stress signal transduction pathways. Such proteins can be manipulated, for example, to protect cells against stress due to disease or environmental conditions.
It is an object of the invention to provide the amino acid sequence and DNA coding sequence of a unique member of the MAP kinase kinase family. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is an isolated and purified human mitogen-activated protein kinase kinase 7 (MKK7) protein having an amino acid sequence which is at least 99% identical to SEQ ID NO:2. Percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
Still another embodiment of the invention is an MKK7 fusion protein comprising a first protein segment and a second protein segment fused together by means of a peptide bond. The first protein segment consists of a human MKK7 protein as shown in SEQ ID NO:2.
Even another embodiment of the invention is a cDNA molecule which encodes a human MKK7 protein having an amino acid sequence which is at least 99% identical to SEQ ID NO:2. Percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
Another embodiment of the invention is a cDNA molecule which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO:1. Percent identity is determined using a Smith-Waterman homology search algorithm as implemented in a MPSRCH program using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
A further embodiment of the invention is an isolated and purified subgenomic polynucleotide comprising a nucleotide sequence which hybridizes to SEQ ID NO:1 after washing with 0.2xc3x97SSC at 65xc2x0 C. The nucleotide sequence encodes a human MKK7 protein having the amino acid sequence of SEQ ID NO:2.
Still another embodiment of the invention is a construct comprising a promoter and a polynucleotide segment encoding a human MKK7 protein as shown in SEQ ID NO:2. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter.
Yet another embodiment of the invention is a host cell comprising a construct which comprises a promoter and a polynucleotide segment encoding a human MKK7 protein as shown in SEQ ID NO:2.
A further embodiment of the invention is a recombinant host cell comprising a new transcription initiation unit. The new transcription initiation unit comprises in 5xe2x80x2 to 3xe2x80x2 order an exogenous regulatory sequence, an exogenous exon, and a splice donor site. The new transcription initiation unit is located upstream of a coding sequence of an MKK7 gene as shown in SEQ ID NO:1. The exogenous regulatory sequence controls transcription of the coding sequence of the MKK7 gene.
Even another embodiment of the invention is a polynucleotide probe comprising at least 12 contiguous nucleotides of SEQ ID NO:1 and a detectable label.
Another embodiment of the invention is a method of increasing transcription of a gene responsive to an AP-1 transcription factor. A cell is contacted with a polynucleotide encoding all or a portion of an MKK7 protein under conditions where said all or a portion of the MKK7 protein phosphorylates a c-Jun NH2 terminal kinase. Phosphorylation of the c-Jun NH2-terminal kinase thereby phosphorylates an AP-1 transcription factor. Transcription of the gene is thereby increased.
Still another embodiment of the invention is a method of decreasing transcription of a gene responsive to an AP-1 transcription factor. A cell is contacted with a polynucleotide encoding a reagent which binds to an MKK7 gene or expression product. The ability of MKK7 protein to phosphorylate a c-Jun NH2-terminal kinase is thereby decreased.
Yet another embodiment of the invention is a method of inducing apoptosis of a cell. A cell is contacted with a polynucleotide encoding all or a portion of an MKK7 expression product. The all or a portion of the MKK7 expression product is capable of inducing apoptosis of the cell. Apoptosis of the cell is thereby induced.
Even another embodiment of the invention is a method of preventing apoptosis of a cell. A cell is contacted with a reagent which binds to an MKK7 gene or expression product. Apoptosis of the cell is thereby prevented.
A further embodiment of the invention is a method of identifying a test compound which modulates signal transduction through a JNK pathway. A biological sample is contacted with a test compound. Phosphorylation or synthesis of an MKK7 protein ash shown in SEQ ID NO:2 or phosphorylation of a substrate of the MKK7 protein is detected. A test compound which increases or decreases phosphorylation or synthesis of the MKK7 protein or phosphorylation of the MKK7 substrate identifies a potential drug for modulating signal transduction through the JNK pathway.
The present invention provides the art with the amino acid sequence and DNA coding sequence of human MKK7, a unique member of the MAP kinase kinase family. The invention can be used, inter alia, to regulate gene transcription and to effect or prevent apoptosis.